Isolation of bacterial luciferase

ABSTRACT

The present invention provides a novel means for the isolation of bacterial luciferase and a novel affinity resin useful in said isolation.

The research described herein was supported in part by grants from theNational Science Foundation (PCM 79-25335).

This application is a continuation-in-part of U.S. application Ser. No.230,178, filed Jan. 30, 1981, now abandoned.

BACKGROUND OF THE INVENTION

The generally practiced method for the isolation of bacterial luciferaseinvolving batch adsorption technique is described by J. WoodlandHastings. et. al., in Methods in Enzymology, Vol. LVII, 135 (1978) andA. Gunsalus-Miguel, et. al., J. Biol. Chem. 247(2), 398-404 (1972).Isolation of bacterial luciferase by affinity chromatography usingimmobilized flavin mononucleotide to bind the enzyme is described by C.A. Waters, et. al., in Biochem. Biophys. Res. Comm. 57, No. 4, 1152(1974). Neither of these methods is particularly efficient in that eachinvolves a considerable amount of time to perform and/or low yields ofenzyme are recovered.

The method of isolating bacterial luciferase as described herein notonly reduces significantly the amount of time required to isolate theenzyme, but also results in high yields of relatively pure enzyme.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for isolating ofpurifying bacterial luciferase which comprises suspending the samplecontaining the enzyme to be isolated in an anionic buffer, contactingsaid suspended sample with an affinity resin, described below, washingthe mixture with the anionic buffer followed by application of acationic buffer to release the enzyme which may then be isolated.

Another embodiment of the present invention is a novel affinity resinwhich comprises a support material to which is attached a spacer armwhich in turn is attached to a ligand which specifically binds thebacterial luciferase. Schematically the affinity resin may berepresented as follows:

    support material˜spacer˜ligand                 Formula I.

Suitable support material which may also be referred to as gel beads orresins, are any polysaccharide base matrix, such as sepharose, sephadex,cellulose; or, an acrylamide base or polyacrylamide which also may becross-linked with a sepharose or sephadex or a polyacrylonitrile basesuch as the particulate support material described in U.S. Pat. No.4,143,203; or, nylon surfaces, such as, nylon fibers or mesh; or, glasssurfaces, such as, glass beads and in particular controlled-pore glass(CPG) as described by H. H. Weetall and A. M. Filbert, Meth. Enzymol.34, 59-72 (1974).

The spacer arm is incorporated into the affinity resin by reaction ofthe support material with any of a variety of conventional difunctionalcompounds such as alkylenediamines, aliphatic aminoacids or a diglycidylether, and the ligand is a compound which binds to bacterial luciferase.

DETAILED DESCRIPTION OF THE INVENTION

Suitable support material for use in preparing the affinity resin of thepresent invention bear groups which will react with the difunctionalcompound utilized to incorporate the spacer arm into the affinity resinor are capable of being rendered reactive to said compounds. Suitablereactive groups which may be present on the support material includecarboxy, hydroxy, sulfhydryl, amino, epoxy or carboxysuccinimide groups.It is important that the support material not interact with the proteinbeing isolated, i.e., the bacterial luciferase. When nylon is employedas the support material it is treated, for example, withN,N-dimethyl-1,3-diaminopropane by the general procedure described by W.E. Hornby, et. al., FEBS Letters 23, 114 (1972). Succinimidederivitization of resins bearing carboxy groups may be accomplished bytreatment with N-hydroxylated succinimide using carbodiimide activation.When CPG is the support material the functional OH is a silanol which istreated with an organosilane such as epoxysilane, sulfhydrylsilane,alkyl-C₁₋₄ -amine silane or an alkyl C₁₋₄ -halosilane such asalkylchlorosilane by generally known procedures; see H. H. Weetall andA. M. Filbert, ibid.

As a matter of convenience the affinity resin of the present inventionis represented schematically as in Formula I and the manner ofconstructing or preparing the affinity resin is set forth in terms ofbeginning with a support material and reacting it with a difunctionalcompound to give a support material˜spacer arm unit which in turn isreacted with the ligand. However, it will be readily apparent that theaffinity resin may be constructed by reacting the ligand with thedifunctional compound to give a spacer arm˜ligand unit which in turn isreacted with the support material. Also commercially available resins orcoupling gels having the spacer arm already attached, such as,AH-Sepharose 4B and CH-Sepharose 4B, may be utilized in preparing theaffinity resin of the present invention.

Difunctional compounds which are used to provide the spacer arm moietyof the affinity resins of the present invention are represented by thefollowing Formulas II and III: ##STR1## In Formula II, Q is a straightor branched alkylene moiety having from 2 to 8 carbon atoms, and R isamino, carboxy or carboxysuccinimide. In Formula III, m is an integerfrom 2 to 6.

Carboxysuccinimide is taken to mean the group: ##STR2##

In Formula II it is preferred that Q is a straight chain alkylenemoiety, i.e., --(CH₂)--₂₋₈ although branching of 1 or 2 carbons issuitable for the present invention. The compounds of Formulas II and IIIare commercially available or can be prepared by procedures well knownin the art. Illustrative examples of compounds of Formula II areethylenediamine, 1,3-propylenediamine, 1,6-hexamethylenediamine,1,3-isobutylenediamine, 1,4-butylenediamine, glycine, β-alanine,alanine, α-aminopropionic acid, γ-aminopropionic acid, γ-aminobutyricacid, γ-aminohexanoic acid and of course the correspondingcarboxysuccinimide derivatives thereof obtained via reaction withN-hydroxysuccinimide. Illustrative examples of compounds of Formula IIIare 1,4-bis-(2,3-epoxypropoxy)butane, 1,2-bis(2,3-epoxypropoxy)ethane,and 1,6-bis(2,3-epoxypropoxy)hexane.

The ligand portion of Formula I are compounds which have a specificaffinity for bacterial luciferase. In addition to2-(2,4-dichloro-6-phenylphenoxy)ethylamine and(2-(2,3-dichloro-6-phenylphenoxy)ethylamine typically the ligandsemployed in the present invention are diphenylalkylene or ortho, meta-or para-biphenylylalkylene containing compounds of the following FormulaIV. ##STR3## wherein X is --COOH, --COhalogen, halogen, OH, SH, NH₂ orepoxy, i.e., ##STR4## Y is a bond or an alkylene chain of 1 to 4 carbonatoms, one or two carbon atoms of which may be branched; Z is hydrogenor a straight or branched alkyl group having from 1 to 4 carbon atoms; qis zero or one; p is zero, one or two; R₁ is hydrogen, phenyl or phenylsubstituted with one or two substituents selected from halogen,trihalomethyl, e.g., trifluoromethyl, a straight or branched alkoxygroup having from 1 to 4 carbon atoms or a straight or branched alkylgroup having from 1 to 4 carbon atoms; and R₂ is ortho-, meta-, orpara-biphenylyl, phenyl, or phenyl substituted with one or twosubstituents selected from halogen, trihalomethyl, e.g.,trifluoromethyl, a straight or branched alkoxy group having from 1 to 4carbon atoms or a straight or branched alkyl group having from 1 to 4carbon atoms with the provisos that: (a) when R₂ is biphenylyl each of pand q is zero and R₁ is hydrogen; (b) when p is two, q is zero; and (c)when R₂ is other than biphenylyl, R₁ is other than hydrogen.

As used herein halo and halogen means chloro, fluoro, bromo and iodo.Preferred halogens are chloro and bromo.

Illustrative examples of alkylene groups which Y represents areethylene, 1,3-propylene, 1,2-isopropylene and 1,4-butylene.

Illustrative examples of alkyl groups which Z represents or which may bepresent as substituents on the phenyl rings represented by R₁ and R₂ aremethyl, ethyl, n-propyl, isopropyl and n-butyl.

Illustrative examples of alkoxy groups which may be present on thephenyl rings represented by R₁ and R₂ are methoxy, ethoxy, andn-propoxy.

Ligands of Formula IV wherein X is COOH, COhalogen and NH₂ arepreferred. Ligands of Formula IV wherein R₁ and R₂ are phenyl or whereinZ is hydrogen, methyl or ethyl are also preferred. The most preferredligands of Formula IV are 2,2-diphenylpropylamine,1,1-diphenylpropylamine, 3,3-diphenylpropylamine, 2,2-diphenylpropionicacid and 3,3-diphenylpropionic acid.

When Y in Formula IV contains at least three carbon atoms in a straightchain relationship, i.e., Y is 1,3-propylene or 1,3-isobutylene, theligand may be attached directly to the support material bypassing theuse of a spacer arm to give an affinity resin which may be depictedschematically as

    support material˜ligand                              Formula V

wherein support material and ligand are as defined hereinabove except Ycontains at least 3 straight chain carbon atoms.

It should be recognized that as the term ligand is used in reference toaffinity resin the terminal functional group represented by X in FormulaIV has been reacted with the functional group carried on the supportmaterial in the case of affinity resins of Formula V or with the distalend, i.e., end furthest from the support material, of the spacer arm inthe case of affinity resins of Formula I.

The compounds of Formulas I and II are reacted with suitable supportmaterials by procedures well known in the art. In preparing the affinityresin of Formula I, the reaction of appropriate support materials asdescribed hereinbelow with the compounds of Formulas II and III asdescribed above will result in support material-spacer arm units havingthe general structures depicted in Formulas A to F set forth in Chart A.In Formulas A to F, O˜ represents the support material; the groups R andQ have the meanings defined in Formula II; m has the meaning defined inFormula III; R₃ is oxygen or sulfur; and R₄ is oxygen, sulfur or --NH--.Illustratively, support materials bearing hydroxyl or sulfhydryl groupsare allowed to react with compounds of Formula II by the proceduresdescribed by J. Porath, Nature 218, 834 (1968) and J. Porath, et. al.,J. Chromatogr. 86, 53 (1973) whereby cyanogen bromide is suspended in anaqueous alkaline solution, e.g., 0.1 M sodium bicarbonate, having a pHof 12, to which is added the support material, such as agarose, at aratio of 2 ml of cyanogen bromide solution per each ml of resinsuspended in 0.1 M aqueous sodium bicarbonate. The mixture is incubatedabout ten minutes with gentle stirring at room temperature, vacuumfiltered then washed with several volumes of cold distilled water toneutrality to give activated support material suitable for coupling withcompounds of Formula II.

A compound of Formula II is added to a 0.2 M aqueous sodium bicarbonatebuffer (pH 9) at a concentration of 5 to 50 mg of compound per ml ofbuffer. The buffer solution is added to a sufficient quantity of theactivated support material described above to give a 1:2 dilution ofcompound to resin, and the mixture is incubated at 4° C. for 12 to 24hours. See Formula A of Chart A.

In coupling the above-described hydroxy containing resin or gel, i.e.,support material with a compound of Formula III, the support materialneed not be activated and is suspended in 0.6 M aqueous sodium hydroxidecontaining 2 mg of sodium borohydride per mil of solution. Equal volumesof the thus suspended resin and a compound of Formula III are reacted atroom temperature (about 25° C.) with gentle stirring for 12 to 24 hoursthen washed with distilled water to pH 7. See Formula B wherein R₄ isoxygen or sulfur. Note that the --OH may be in the penultimate positionwith the attachment to the terminal carbon, as depicted, or theattachment may be to the penultimate carbon giving the primary alcohol.

Support materials bearing amino groups may be coupled to compounds ofFormula II wherein R is carboxy or carboxysuccinimide or to compounds ofFormula III, all reactions being carried out by procedures known in theart. For example, in coupling an amino bearing support material to anaminocarboxylic acid of Formula II, an excess of the acid is combinedwith the support material in a protic solvent, such as, aqueousdimethylformamide, at a pH of about 4.7 in the presence of a 10 to 100fold molar excess of a water soluble carbodiimide, such as,N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide HCl. See Formula C. Incoupling a succinimide derivative of Formula II with an amino bearingsupport material a 10-fold molar excess of succinimide derivative isadded to the support material in an aqueous buffer system, such as, 0.2M triethylamine acetate or phosphate or pyrophosphate at a pH rangingfrom 7 to 9.5 and the mixture is stirred at room temperature for about12 to 24 hours. See Formula C. The compounds of Formula III are coupledto support materials bearing amino functions by combining an excess ofthe Formula III compound with the support material in a 50% aqueoussolution of dioxane at a pH of about 11. The reaction is allowed toproceed for about 12 hours at 50° C. or up to 24 hours at roomtemperature. See Formula B wherein R₄ is NH. Under essentially the sameconditions support materials bearing a halogen function are coupled tocompounds of Formula II. See Formula D.

Support materials bearing carboxy groups may be coupled to compounds ofFormula II by the same general carbodiimide activation proceduredescribed hereinabove for coupling amino bearing support materials tothe aminocarboxylic acids of Formula II. See Formula E. Supportmaterials bearing succinimide groups may be coupled to compounds ofFormula II by the same general procedures described hereinabove forcoupling amino bearing support materials with compounds of Formula IIwherein R is a carboxysuccinimide group. See Formula E. Also supportmaterials bearing functional epoxy groups are coupled to compounds ofFormula II by the same general procedure as described for coupling aminobearing support materials to compounds of Formula III. See Formula F.

When compounds of Formula III are coupled to appropriate supportmaterials, i.e., those bearing either functional amines, hydroxyl orsulfhydryl groups, under conditions of lower pH than described above,the epoxide ring opens in such a manner as to have a supportmaterial-spacer arm unit having a primary alcohol as depicted in FormulaG wherein R₄ is oxygen, sulfur or --NH-- and m is an integer of 2 to 6.Similarly reaction of compounds of Formula II with support materialsbearing an epoxy function under conditions of lower pH results insupport material-spacer arm units as depicted in Formula H wherein Q andR have the meanings defined in Formula II.

The thus formed units depicted in Formulas A to H are reacted with theligands of Formula IV by known means as generally described above. Thecompound units of Formulas A, C-F and G containing a terminal aminegroup are reacted with compounds of Formula IV wherein X is COOH and thecompounds of Formulas A, D-F and H containing a terminal carboxy arereacted with compounds of Formula IV wherein X is NH₂ or with2-(2,4-dichloro-5-phenylphenoxy)ethylamine or2-(2,3-dichloro-6-phenylphenoxy)ethylamine by the standard carbodiimideactivation coupling reaction described above. The compound units of A,D-F and H containing a terminal carboxysuccinimide group are reactedwith a ligand of Formula IV wherein X is NH₂ or with2-(2,4-dichloro-5-phenylphenoxy)ethylamine or2-(2,3-dichloro-6-phenylphenoxy)ethylamine by combining the appropriatecompound unit with a 10-fold molar excess of ligand in an aqueous buffersystem, e.g., 0.2 M triethylamine acetate or a phosphate orpyrophosphate buffer at a pH of about 7 to 9.5 at about 25° C. withstirring for about 12 to 24 hours. The compounds of Formulas B and G arereacted with the ligands of Formula IV wherein X is NH₂, sulfhydryl orhydroxyl or with 2-(2,4-dichloro-5-phenylphenoxy)ethylamine or2-(2,3-dichloro-6-phenylphenoxy)ethylamine by combining the ligand andan excess of the epoxide compound of Formula B or G in, for example, a50 percent aqueous dioxane solution at a pH of about 11 and allowing thereaction to proceed at about 50° C. for about 12 hours. In a similarmanner the compounds of Formulas A, C- F and H containing a terminalamine group and the ligands of Formula IV wherein X is epoxy or halo or##STR5## are coupled and when X is halo-containing it may be desirableto run the reaction at about 25° C. for up to about 24 hours.

The thus formed affinity resins may be depicted as shown in Formulas Jand K. In Formula J, O˜ represents the support material which hasattached thereto one of the functional linkages depicted in theleft-hand bracket which linkage is attached to one end of the group Qwhich is defined as in Formula II. The other end of the group Q isattached to one of the functional linkages in the right-hand bracketwhich linkage is attached to L. The group L in Formulas J and K is thesame as the compounds of Formula IV only the X group of the Formula IVcompounds is removed, i.e., L is ##STR6## wherein Y, Z, q, p, R₁ and R₂are as defined in Formula IV, or when the functional linkage in theright-hand bracket is other than ##STR7## L may be2-(2,4-dichloro-6-phenylphenoxy)ethanyl or2-(2,3-dichloro-6-phenylphenoxy)ethanyl.

In the affinity resins depicted by Formula K the support material O˜ isattached to one of the functional linkages in the bracket which linkageis attached to the remainder of the molecule via --CH₂ --. In Formula K,m is the integer 2 to 6, each of R₄ and R₅ is sulfur, oxygen or NH and Lhas the meaning defined in Formula J with the proviso that when L is2-(2,4-dichloro-6-phenylphenoxy)ethanyl or2-(2,3-dichloro-6-phenylphenoxy)ethanyl, R₅ is NH.

The affinity resins of Formula V are prepared by reacting a compound ofFormula IV wherein Y contains at least 3 carbons in a straight chainrelationship with suitable support materials utilizing the same chemicalprocedures described hereinabove for preparing the affinity resinsdepicted in Formulas J and K. The resulting products are illustrated byFormula M wherein R₆ is NH, sulfur or oxygen and L represents thecompounds of Formula IV except the X group is removed and Y contains atleast three straight chain carbon atoms.

Most of the compounds of general Formula IV are known in the art and areeither commercially available or are prepared by generally knownmethods, and it is apparent that certain of the compounds of Formula IVcan be used to prepare other compounds of said formula.

Compounds wherein Y is methylene and both p and q are zero may beprepared by alkylating for example an appropriately substituteddiphenylacetonitrile with an alkylC₁₋₄ halide in a solution ofdimethylformamide and sodium hydride then reduced with, e.g., lithiumaluminum hydride to give the correspondingly substituted2-alkyl-2,2-diphenylethaneamine. The amines may also be obtained byconverting the alkylated nitrile to the corresponding amide by treatmentwith sulfuric acid with subsequent reduction of the amide using, e.g.,borone. Or, the alkylated nitrile can be reduced with, e.g.,diisobutylaluminum hydride to give the corresponding carboxaldehydederivative which may be converted to the corresponding epoxide using atrimethylsulfonium halide. Also, the nitrile may be converted to thecorresponding carboxylic acid by treatment with aqueous base andtreatment of the acid with thionyl halide, e.g., thionyl chloride, givesthe corresponding acid halide. Or the acid may be treated with a loweralcohol such as ethanol under mildly acidic conditions to give the loweralkyl ester which in turn is reduced with, e.g., lithium aluminumhydride to give the corresponding alcohol. The thus obtained alcohol maybe treated with P₂ S₅ to give the corresponding thiol, or, the alcoholthus obtained may be treated with a thionyl halide such as thionylchloride or with phosphonyl chloride/phosphorus pentachloride incarbontetrahalide such as carbon tetrachloride and triphenylphosphine togive the corresponding halide derivative. The resultant halide may betreated with potassium cyanide to give the correspondingly substituted2-alkyl-2,2-diphenylethanonitrile which may be treated as describedhereinabove to give compounds of Formula IV wherein Y is ethylene and byfollowing this procedure all of the compounds wherein Y is an alkylenechain of 1 to 4 carbon atoms and both p and q are zero are prepared.Compounds of Formula IV wherein p and q are other than zero can beprepared in a similar manner from the appropriate nitrile which may beprepared by known procedures. For example, the nitrile may be obtainedby treating an appropriately substituted ketone derivative, e.g., benzylphenyl ketone, phenethyl phenyl ketone or benzophenone with an alkylGrignard to give the corresponding alkylated alcohol which can beconverted tothe corresponding thiol using P₂ S₅ or to the corresondinghalide using in carbon tetrahalide, e.g., tetrachloride andtriphenylphosphine with alcohol. The halide can then be used to obtainthe nitrile by treating with potassium cyanide and dimethylsulfoxidewhich can be used to obtain the other compounds of Formula IV asdescribed above. However, in utilizing such a nitrile to give the aminederivative it may be more desirable to first hydrolyze the nitrile tothe amide using sulfuric acid then treat the amide with Br₂ /NaOH underthe conditions of a Hofman Reaction [Ber. 14, 2775 (1881)] to give theamine.

The thus formed novel affinity resins specifically bind bacterialluciferase, and in particular luciferases from samples of lysed Vibriofischeri, and Photobacterium phosphoreum and Vibrio harveyi bind to andelute from the affinity resins resulting in high yields of pure enzyme.The means by which this binding is believed to occur is discussed inBiochemistry 1981, 20, 5524-5528. See also Biophysical Journal 33, part2, 255 (1981). Another important aspect of the present invention is thefact that the amount of time required to recover these high yields ofhomogeneous enzyme is reduced quite significantly from previously knownprocedures. Another marked advantage of the presently claimed method isthat it can be scaled up or down with relative ease to accommodaterecovery of large or small quantities of enzyme as needed. Additionally,the novel affinity resins of the present invention may be usedrepeatedly for isolation of enzyme. That is, after the affinity resinhas been used to isolate bacterial luciferase, it may be washed with,e.g., a 1:1 mixture of 8 M urea and 50% ethanol containing, 0.08 Mphosphate, then reequilibrated with anionic buffer and used again forisolation of enzyme.

Bacterial luciferase is known to be useful in the assay of proteases asthe luciferase enzyme is highly susceptible to inactivation by a widevariety of proteases such as, for example, trypsin, elastase,chymotrypsin, plasminogen and collagenase, several of which are known tobe involved or are implicated in certain disease conditions, such as,autoimmune diseases, e.g., rheumatoid arthritis, in certain types ofcarcinoma, emphysema and in peridontal diseases. Therefore, the use ofbacterial luciferase in an enzyme assay system provides a useful meansof detecting protease activity which may lead to the detection ofcertain disease conditions. Aside from its utility in assayingproteinases, luciferase can also be used in a coupled enzyme assaysystem for detecting the levels of nucleotides like NADH, NADPH, and FMNin serum or tissue samples. See P. E. Stanley (1978) Meth. Enzymol. 57,215-223, or for detecting the presence of other enzymes utilizing thesenucleotides, see P. E. Stanley (1978) Meth. Enzymol. 57, 181-189.

When isolating the bacterial luciferase enzyme by means of the presentinvention, in general the novel affinity resin is equilibrated with ananionic buffer solution and is then contacted with a sufficient amountof enzyme sample suspended in the same anionic buffer to saturate thebinding sites on the affinity resin, after which the affinity resin iswashed with additional anionic buffer to remove materials contained inthe enzyme sample which do not bind and subsequently the affinity resinis eluted or washed with a cationic or neutral buffer to release thebound enzyme. Thus the enzyme may be isolated by any means which willpermit contact of the sample containing the bacterial luciferase to beisolated with the equilibrated affinity resin in an appropriate anionicbuffer with subsequent washing of the affinity resin containing thebound enzyme followed by treatment with an appropriate cationic orneutral buffer. Thus the enzyme may be isolated using batch isolationtechniques or column chromatographic techniques, the latter of which ispreferred. It is important, particularly when column chromatographictechniques are employed, that the affinity resin be saturated with theenzyme sample. When using batch separation methods binding to theaffinity resin is measured by disappearance of enzyme activity from thesuspending solution, and following the removal of unbound protein fromthe mixture, release and recovery of the enzyme by treatment withcationic buffer is measured by the increase in enzyme activity in thesupernatant. Similarly when using column chromatography, saturation ofthe affinity resin is measured by the appearance of enzyme activity inthe eluate fractions collected.

It has been determined that the enzyme binding capacity of the affinityresin is about 5 to 25 mg of bacterial luciferase per milliliter (ml) ofsuspended affinity resin depending upon the number of affinity ligandmolecules attached to the support resin. When using columnchromatography the capacity of column containing the equilibratedaffinity resin is about 5 to 25 mg of bacterial luciferase per ml ofcolumn volume.

The enzyme may be isolated at various temperatures although it isconvenient and practical to perform the isolation in a cold room, e.g.,about 4° C. Of course, the separation could be performed atsignificantly higher or lower temperatures, however, it may becomenecessary to add to the medium agents which will retard bacterial growthand, of course at very low temperatures, means to prevent freezing ofthe medium may be required.

It is known that bacterial luciferase has an anionic binding site. See,e.g., Holzman and Baldwin, Biochem. Biophys. Res. Comm. 94, No. 4, 1199(1980) and Proc. Natl. Acad. Sci. 77, No. 11, 6262 (1980). Therefore ananionic buffer system is employed in the present invention to facilitatebinding of the enzyme to the affinity resin. Generally the enzymeaffinity for the novel resin is enhanced with increasing anionicstrength of the buffer. The anionic buffer has a pH ranging from about6.5 to 9.5, and an overall ionic strength of about 0.01 M to about 2 M.Preferably the pH of the buffer is about 8 to 8.5 and the ionic strengthis about 0.2 to 1 M. The anionic buffer consists of an aqueous solutionof an alkali metal salt, e.g., potassium or sodium, of phosphate,preferably, although salts of citrate, pyrophosphate, arsenate, sulfateor equivalents thereof may be used or mixtures of these salts may beused. The anionic buffer also contains a thiol containing reducing agentat a concentration of about 1 mM. Suitable reducing agents includedithioerythritol, dithiothrietol, mercaptoethanol, cysteamine, cysteine,thiodiglycol, or glutathione.

As indicated hereinabove the anionic buffer is used to equilibrate theaffinity resin, and is also used to suspend the sample containing thebacterial luciferase to be isolated and is used to wash the unboundmaterials away from the affinity resin once it becomes saturated withenzyme.

The cationic or neutral buffer employed in the present invention reducesthe affinity of the enzyme for the novel resin. Generally any organicamine containing buffer may be used, such as commercially availableTRIS, 2-amino-2-hydroxymethyl-2,4-propanediol; HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TES,2-[[tris-(hydroxymethyl)methyl]amino]ethanesulfonic acid, orethanolamine alone or in combination with other amine buffers.Ethanolamine is particularly useful in the isolation of Vibrio harveyiluciferase. The ionic strength of the buffer should be relatively lowand not exceed about 1 M. The pH should be maintained at about 6.5 to9.5. The cationic buffer system also contains a thiol containingreducing agent at a concentration of about 1 mM. Suitable reducingagents include those enumerated above in conjunction with the anionicbuffer system.

Typically in practicing the invention an affinity resin as describedherein is suspended in an anionic buffer as described above and pouredinto a column. The resin is equilibrated with the anionic buffer. Thesample containing the bacterial luciferase to be isolated is suspendedin the anionic buffer and poured onto the column. The column size isdetermined by the amount of enzyme present in the sample to be isolatedwhich is determined by the enzyme activity of the sample. Once theaffinity resin is saturated the column is washed with anionic buffer toremove unbound material, then the column is eluted using cationic orneutral buffer above described. The elution step generally requiresabout 4 to 8 column volumes of buffer. Fractions are collected and anelution curve is generated. The yield of enzyme recovered is measured bythe enzyme activity recovered.

If all fractions collected are pooled, essentially 100 percent of theenzyme is recovered. We have found that when 90% of the initial total ofenzyme activity is recovered the protein or enzyme is about 50% pure. Ofcourse one may select only those fractions having highest enzymeactivity to increase the purity of recovered protein. When 75 percent ofthe initial total activity is recovered the purity of the enzyme isabout 65% and when 50% of the initial total activity is recovered theenzyme is about 90% pure.

The following examples further illustrate the invention.

EXAMPLE 1

The following illustrates the preparation of a typical affinity resin asdepicted by Formula K wherein the bracketed functional group is ##STR8##and R₄ is oxygen, m is 4, R₅ is NH and L is 2,2-diphenylpropanyl.

To about 50 ml of Sepharose 6B is added 50 ml of 1,4-diglycidyl ether,i.e., 1,4-bis(2,3-epoxypropoxy)butane, 50 ml of 0.6 M sodium hydroxideand 100 mg of sodium borohydride. The slurry thus formed is mixed byrotation for about 8 hours at about 25° C., then poured onto a sinteredglass funnel and washed to neutrality with distilled water and suctiondried. This procedure is generally described by Sunderberg and Porath,J. Chrom. 90, 87 (1974).

The thus formed epoxy activated Sepharose is reacted with2,2-diphenylpropylamine. For each gram of epoxy activated Sepharoseemployed 100 mg of 2,2-diphenylpropylamine is used. The alkylamine isdissolved in 50% dioxane/50% 0.20 M carbonate buffer pH 10.5 (1 ml ofthe liquid in dioxane buffer per gram of Sepharose). The solution pH ismaintained at pH 10.5. The activated Sepharose is then added and theflask containing the thus formed slurry is placed in a water bath at˜60° and rotated for 24 hours. During the first 12 hours the pH of theslurry is checked at three hour intervals, then again at 18 hours and 24hours and adjusted to pH 10.5 if necessary. The immobilized inhibitor,that is, the thus formed affinity resin is then carefully washed out ofthe flask and into a coarse pore sintered glass funnel attached to avacuum filtration system. For each gram of the suction-dried Sepharoseused initially, the affinity resin is washed sequentially with thefollowing mixtures: 6 ml of 1:1 dioxane/water, 6 ml of 1:1 dioxane/0.20M phosphate pH 7.0 and 12 ml of 95% ethanol. The affinity resin may beused directly or may be stored in 50% ethanol/water. Generally, 1-2μmole of ligand is attached per milliliter of resin by this procedure.

To pack a column, a gel slurry is prepared by adding an equal volume ofapplication buffer, i.e., anionic buffer, and swirling gently. Theslurry is poured into the column. The packed column is equilibrated bywashing with about 4 column volumes of anionic buffer before beginningsample application.

EXAMPLE 2

The following describes the preparation of an affinity resin as depictedby Formula J wherein the left-hand bracketed functional group is##STR9## and R₃ is oxygen: the right-hand bracketed functional group is##STR10## and L is 2,2-diphenylpropanyl.

A 5 ml solution of cyanogen bromide (prepared at 100 mg/ml) indimethylformamide is added to 100 ml of Sepharose 6B which had beenequilibrated in 100 ml of 2 M sodium carbonate. During cyanogen bromideactivation, which requires about 10 minutes and is carried out at about25° C., the gel is held in a sintered-glass funnel and is gentlyagitated by bubbling nitrogen through the fritted glass disk of thefunnel. The activation procedure is outlined by March, et al., (1974)Anal. Biochem. 60, 149. Following cyanogen bromide activation the gel iswashed with water to neutrality, then resuspended in 200 ml, 0.10 Maqueous solution of 1,6-hexanediamine at pH 9.5. The mixture is allowedto gently stir at 4° C. for 12 hours, then is washed to neutrality withwater on the sintered glass funnel.

To 10 ml of the thus formed resin or gel bead spacer arm unit is added100 mg of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide HCl and 200 mgof 2,2-diphenylpropionic acid in 20 ml of 50% dioxane/water, pH 6.0.Upon completion of the addition the pH is adjusted 6.0 and the mixtureis stirred using a rotary stirring apparatus. After about 2 hours the pHtended to decrease and was adjusted to 6 as needed. After the initialdrop in pH the mixture is stirred for 24 hours at about 25° C. The gelis washed to neutrality with water on a vacuum filter to give the novelaffinity resin.

The foregoing carbodiimide coupling typically results in substitution inthe range of 1 to 2μ moles per ml of resin.

EXAMPLE 3

The following illustrates the method of isolating bacterial luciferasefrom a sample as herein claimed. The enzyme source is Vibrio harveyi.

A cell lysate is prepared from 100 gm of frozen cell paste by theosmotic lysis technique described by Hastings, et al., Meth. Enzymol.57, 135 (1978). Ammonium sulfate is added to the lysate (1100 ml) to 40%of saturation and centrifuged at 8000 RPM for 1-1.5 hours in a SorvallGSA rotor at 4° C. The lysate is allowed to equilibrate with theammonium sulfate for about 30 minutes prior to centrifugation. Thesupernatant (1100 ml) is collected and more ammonium sulfate added to80% saturation. The pellets from the first centrifugation, containingcell debris and precipitated protein, are discarded. The supernatantwith ammonium sulfate at 80% saturation is centrifuged at 8000 RPM, 4°C., for one hour in a GSA rotor. The pellets are collected andtransferred to dialysis tubing and dialyzed at 4° C. into theapplication or anionic buffer. Transfer of the pellets into the dialysistubing, preparation of the buffer for dialysis, etc., requires about 2hours. The crude luciferase sample is dialyzed versus 3 changes of 1liter each for a total of about 12 hours at 4° C. The sample is thencentrifuged at 15,000 RPM in an SS-34 rotor for 30 minutes at 4° C. Thetotal volume of supernatant is 245 ml. Half of the clear solution (120ml) is then applied to a 15 ml column of the affinity matrixequilibrated in the application buffer. The application flowrate isabout 40 ml/hr. By assays of eluate from the column for luciferaseactivity, it is determined that the column is saturated with luciferaseby this amount of activity. The column is then washed with 4 columnvolumes (60 ml) of application buffer at a flowrate of about 15 ml/hr toelute unbound or weakly bound protein. The column is then eluted with 4column volumes of elution buffer; 1 ml fractions are collected andassayed for luciferase activity. Fractions containing about 90% of theluciferase activity are pooled, concentrated by ammonium sulfateprecipitation (80% of saturation), dialyzed and subjected tochromatography on aminohexyl Sepharose as described by Hastings, et al,ibid.

Purity of the enzyme is estimated by Coomassie blue staining of proteinbands resolved by sodium dodecylsulfate gel electrophoresis inpolyacrylamide as described by Laemmli, Nature 227, 680 (1970). Pureluciferase (≧95%) has a specific activity of about 30,000 (±10%) lightunits per mg of protein when assayed by the standard flavin injectionassay described in detail by Hastings, et al. One light unit is1.06×10¹⁰ quanta/sec based on the liquid light standard of Hastings andWeber, J. Opt. Soc. Am. 53, 1410 (1963).

The following Table summarizes the foregoing and the results obtained.

Following the same general procedure as described in Example 3 onlyusing from 2 to 5 grams of cell paste prepared from Vibrio(photobacterium) fischeri or Photobacterium phosphoreum and adjustingthe volumes of reagents appropriately essentially all the appliedaffinity (>95%) is recovered with a purity ≧95%.

                                      TABLE I                                     __________________________________________________________________________    Purification Table for V. harveyi Luciferase                                                              Total Specific                                                                            Fold             % Yield                        Vol.    Total                                                                             Activity*                                                                           Activity                                                                            Activity                                                                            Purification     (Initial             Step      (ml) A.sub.280                                                                        A.sub.280                                                                         (L.U./ml)                                                                           (L.U.)                                                                              L.U./A.sub.280                                                                      (Previous Step)                                                                       Time/Step                                                                              Total                __________________________________________________________________________                                                             Act.)                Osmotic   1100 28.0                                                                             30800                                                                              5300 5.85 × 10.sup.6                                                                189  --         --    --                   Lysis                                           ˜1 hour from                                                            thawed cells                  40% Ammonium                                                                            1100 -- --   5300 5.85 × 10.sup.6                                                               --    --         --    --                   sulfate                                         ˜2 hour cen-            supernatant                                     trifugation                                                                   @ 8000 RPM                                                                    GSA Rotor, 4° C.       80% AMS PPT                                     1-3 hour cent.,                                                               4° C. 8000 RPM                                                         GSA Rotor                     Dialysis into                                                                           245  42.5                                                                             10412                                                                             23900 5.85 × 10.sup.6                                                                562  2.98    12 hours,                                                                              100%                 affinity col-                                   changes of                    umn applica-                                    buffer                        tion buffer                                                                   Affinity  24   6.1                                                                               146                                                                              95000 2.28 × 10.sup.6                                                               15600 27.8    Total run                                                                              88%                  column***                                       time ˜12                pool                                            hours                         Applied                                                                       2.66 × 10.sup.6                                                         Tot. L.U.                                                                     AH-Sepharose                                                                            76.6 1.58                                                                              121                                                                              50160 3.84 × 10.sup.6                                                               32000 2.05    Total run                                                                              66%                  column pool:                                    time ˜16                Applied 60 ml**                                 hours                         A.sub.280 = 3.85 in                                                           5.0 mM PO.sub.4,                                                              pH 7.0, 0.50                                                                  mM DTE                                                                        ˜4.9 × 10.sup.6 T.L.U.                                            __________________________________________________________________________     *1 L.U. = 1.06 × 10.sup.10 hv/sec                                       **Two affinity column preps from the original lysis                           ***Column volume =  15 ml, 2 cm diameter; note only half of total sample      is applied.                                                                   Affinity column application buffer:                                           0.10 M PO (Na/K--Phosphate), pH 8.5.                                          0.50 mM DTE                                                                   0.50 M KCl                                                                    0.50 M NaCl                                                                   Affinity column elution buffer:                                               0.025 M ethanolamine, 5 mM TRIS, pH 9.1.                                      0.50 mM DTE                                                                   Note:                                                                         Both affinity column and AHSeph. column are pumped.                      

    __________________________________________________________________________    CHART A                                                                       __________________________________________________________________________     ##STR11##                    Formula A                                        ##STR12##                    Formula B                                        ##STR13##                    Formula C                                       ○ NHQR                 Formula D                                        ##STR14##                    Formula E                                        ##STR15##                    Formula F                                        ##STR16##                    Formula G                                        ##STR17##                    Formula H                                       __________________________________________________________________________

    ______________________________________                                        CHART B                                                                       ______________________________________                                        Formula J                                                                      ##STR18##                                                                    Formula K                                                                      ##STR19##                                                                     ##STR20##                                                                    Formula M                                                                      ##STR21##                                                                

We claim:
 1. A method for isolating bacterial luciferase which comprisessaturating an affinity resin which comprises a support material, aspacer arm and a ligand as described in the following Formulas J and Kwhereinrepresents the support material; R₃ is oxygen or sulfur; each ofR₄ and R₅ is sulfur, oxygen or --NH--; m is an integer from 2 to 6; Q isa straight or branched alkylene moiety having from 2 to 8 carbon atoms;L is ##STR22## wherein Y is a bond or an alkylene chain of 1 to 4 carbonatoms, one or two carbon atoms of which may be branched; Z is hydrogenor a straight or branched alkyl group having from 1 to 4 carbon atoms; qis zero or one; p is zero, one or two; R₁ is hydrogen, phenyl or phenylsubstituted with one or two substituents selected from halogen,trihalomethyl, a straight or branched alkoxy group having from 1 to 4carbon atoms or a straight or branched alkyl group having from 1 to 4carbon atoms; and R₂ is ortho, meta or para-biphenylyl, phenyl, orphenyl substituted with one or two substituents selected from halogen,trihalomethyl, a straight or branched alkoxy group having from 1 to 4carbon atoms or a straight or branched alkyl group having from 1 to 4carbon atoms with the provisos that: (a) when R₂ is biphenylyl, each ofp and q is zero and R₁ is hydrogen; (b) when p is two, q is zero; and(c) when R₂ is other than biphenylyl, R₁ is other than hydrogen, or whenthe functional linkage in the right hand bracket is other than ##STR23##L is 2-(2,4-dichloro-6-phenylphenoxy)ethanyl or2-(2,3-dichloro-6-phenylphenoxy)ethanyl: ##STR24## with a samplecontaining bacterial luciferase suspended in an appropriate anionicbuffer solution, washing said saturated resin with additional anionicbuffer to remove unbound protein, eluting said washed saturated resinwith an appropriate cationic buffer solution and recovering thebacterial luciferase therefrom.
 2. A method for isolating bacterialluciferase which comprises saturating an affinity resin which comprisesa support material and a ligand as described in the following Formula Mwhereinrepresents the support material; R₆ is --NH--, sulfur or oxygen;L is ##STR25## wherein n' is 3 or 4; Z is hydrogen or a straight orbranched alkyl group having from 1 to 4 carbon atoms; q is zero or one;p is zero, one or two; R₁ is hydrogen, phenyl or phenyl substituted withone or two substituents selected from halogen, trihalomethyl, a straightor branched alkoxy group having from 1 to 4 carbon atoms or a straightor branched alkyl group having from 1 to 4 carbon atoms; and R₂ isortho, meta or para-biphenylyl, phenyl, or phenyl substituted with oneor two substituents selected from halogen, trihalomethyl, a straight orbranched alkoxy group having from 1 to 4 carbon atoms or a straight orbranched alkyl group having from 1 to 4 carbon atoms with the provisosthat: (a) when R₂ is biphenylyl, each of p and q is zero and R₁ ishydrogen; (b) when p is two, q is zero; and (c) when R₂ is other thanbiphenylyl, R₁ is other than hydrogen; ##STR26## with a samplecontaining bacterial luciferase suspended in an appropriate anionicbuffer solution, washing said saturated resin with additional anionicbuffer to remove unbound protein, eluting said washed saturated resinwith an appropriate cationic buffer solution and recovering thebacterial luciferase therefrom.
 3. The method of claim 1 or 2 whereinthe affinity resin is suspended in an appropriate anionic buffer andpoured into a chromatographic column.
 4. The method of claim 3 whereinthe anionic buffer contains sodium phosphate or potassium phosphate. 5.The method of claim 3 wherein the bacterial luciferase source is Vibriofischeri, Photobacterium phosphoreum or Vibrio harveyi.
 6. The method ofclaim 1 or 2 wherein L is 2-(2,3-dichloro-6-phenylphenoxy)ethanyl. 7.The method of claim 1 wherein L has the structure: ##STR27## wherein nis zero to 4; R₁ is hydrogen or phenyl; R₂ is ortho-, meta- orpara-biphenylyl or phenyl; q is zero or one; p is zero, one or two; andZ is hydrogen or a straight or branched alkyl group having from 1 to 4carbon atoms; with the provisos that (a) when R₂ is biphenylyl each of pand q is zero and R₁ is hydrogen, (b) when p is two, q is zero, and (c)when R₂ is other than biphenylyl R₁ is other than hydrogen.
 8. Themethod of claim 7 wherein n is zero to 3; Z is hydrogen, methyl orethyl; and each of R₁ and R₂ is phenyl.
 9. The method of claim 2 whereinL has the structure: ##STR28## wherein n' is 3 or 4; R₁ is hydrogen orphenyl; R₂ is ortho-, meta- or para-biphenylyl or phenyl; q is zero orone; p is zero, one or two; and Z is hydrogen or a straight or branchedalkyl group having from 1 to 4 carbon atoms; with the provisos that (a)when R₂ is biphenylyl each of p and q is zero and R₁ is hydrogen, (b)when p is two, q is zero, and (c) when R₂ is other than biphenylyl R₁ isother than hydrogen.
 10. The method of claim 9 wherein Z is hydrogen,methyl or ethyl, and each of R₁ and R₂ is phenyl.
 11. The method ofclaim 8 or 10 wherein each of p and q is zero, and Z is hydrogen ormethyl.